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Chitosan-Induced Perturbation of Dipalmitoyl-sn-glycero-3-phosphocholine Membrane Bilayer Vincent Chan,*,† Hai-Quan Mao,‡ and Kam W. Leong‡ School of Mechanical and Production Engineering, Division of Thermal and Fluids Engineering and Tissue Engineering Laboratory, Nanyang Technological University, Singapore 639798, Johns Hopkins University School of Medicine, Department of Biomedical Engineering, Baltimore, Maryland 21205, and Johns Hopkins Singapore Pte. Ltd., Singapore 117597 Received December 14, 2000. In Final Form: February 28, 2001 Recently, chitosan, a positively charged polysaccharide in slightly acidic condition, has been used as a membrane perturbant in a novel gene delivery assay. In this study, the fundamental interactions between chitosan and DPPC membrane bilayers were investigated with cross-polarization microscopy, differential scanning calorimetry and Fourier transform (FT) Raman spectroscopy. The cross-polarized images showed that chitosan induced fusions of multilamellar vesicles. It was determined that the mixing of chitosan with dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and subsequent hydration of the mixture at 60 °C significantly suppressed the enthalpy of the gel-liquid crystalline transition in a concentration-dependent manner. Chitosan also affected the thermotropic behavior of DPPC bilayer during the cooling cycle. However, chitosan addition to DPPC had no effect on the main phase transition temperature (Tm) of DPPC bilayers. When DPPC and chitosan were mixed in chloroform before hydration, the initial rate of enthalpy reduction against chitosan concentration was significantly increased. Furthermore, the dependence of the cooperative unit of the DPPCs main transition on the chitosan mole fraction showed that chitosan tuned the intermolecular interactions between neighboring lipid molecules. FT-Raman spectroscopy provided solid evidence that the attractive interchain and intermolecular forces of the hydrophobic core (acyl chains) in the DPPC bilayer were significantly reduced by the chitosan-membrane interactions. The addition of chitosan also reduced the order in the two-dimensional packing of the acyl chains and increased the fluidity of the DPPC bilayer. This study provided new insights into the physicochemical interactions between model membrane and chitosan that might aid the development of a novel membrane perturbant for gene delivery.
Introduction The explosion of genomics information has prompted the development of advanced gene delivery systems for carrying the replacement DNA molecules to cells or tissues.1 To date, most of the ongoing clinical trials of human gene therapy involved the applications of viral or bacterial vectors as carriers of DNA molecules.2 Despite the high transfection efficiency of these viral/bacterial vectors for delivering genes to cells, animal models and humans, the long-term safety, particularly the immunogenicity, of these carriers remains a concern.3,4 Several groups have recently developed new gene delivery systems based on cationic liposomes,5 biodegradable polymers,6 gelatin,7 peptides,8,9 etc. These gene carriers could ef* To whom correspondence may be addressed at Nanyang Technological University, MPE, 50 Nanyang Ave., Singapore 639798. † Nanyang Technological University. ‡ Johns Hopkins University School of Medicine and Johns Hopkins Singapore Pte. Ltd. (1) Ledley, F. D. Curr. Opin. Biotechnol. 1994, 5 (6), 626-636. (2) Tomlinson, E.; Rolland, A. P. J. Controlled Release 1996, 39, 357372. (3) Chen, S. C. J. Virol. 1998, 72, 5757-5761. (4) Gupta, N. Expert Opin. Invest. Drugs 2000, 9 (4), 713-726. (5) Lee, R. J.; Huang, L. Crit. Rev. Ther. Drug Carrier Syst. 1997, 14 (2), 173-206. (6) Boussif, O.; Lezoualch, F.; Zabta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92 (16), 7297-7301. (7) Burgess, D. J. J. Colloid Interface Sci. 1990, 140, 227-239. (8) Gottscahalk, S.; Sparrow, J. T.; Hauer, J.; Mims, M. P.; Leland; F. E.; Woo, S. L.; Smith, L. C. Gene Ther. 1996, 3 (5), 48-57.
fectively encapsulate DNA through electrostatic interactions, deliver the DNA to the nucleus through unknown mechanisms, and occasionally achieve reasonable transgene expression in the target cells/tissues.5-8 One promising class of these carriers is chitosan-DNA nanoparticles synthesized by complexing high-molecular-weight chitosan with plasmid DNA.10-13 The resulting nanoparticles are stable in physiological conditions since chitosan, a polysaccharide derived from crustacean shells, is hydrophobic at neutral pH.13 Chitosan is highly biocompatible, is slowly biodegradable, and has been widely used in controlled drug delivery and weight control therapy.14,15 In a food allergy murine model, it has been shown that the chitosan-DNA nanoparticles can be orally administrated to the animal, transduced gene expression in the intestinal epithelium, and stimulated an immune response to alleviate the anaphylactic responses upon a peanut allergen chal(9) Subramanian, A.; Ranganathan, P.; Diamond, S. L. Nat. Biotechnol. 1999, 17 (9), 873-7. (10) Roy, K.; Mao, H. Q.; Huang, S. K.; Leong, K. W. Nat. Med 1999, 5(4), 387-391. (11) Truong-Le, V. L.; Walsh, S. M.; Schweibert, E.; Mao, H. Q.; Guggino, W. B.; August, J. T.; Leong, K. W. Arch. Biochem. Biophys. 1999, 361 (1), 47-56. (12) Leong, K. W.; Mao, H. Q.; Truong-Le, V. L.; Roy, K.; Walsh, S. M.; August, J. T. J. Controlled Release 1998, 53 (1-3), 183-193. (13) Truong-Le, V. L.; Williams, J. R.; Hildreth, J. E. K.; Leong, K. W. Drug Delivery 1995, 2, 166-174. (14) Bodmeier, R.; Chen, H. G.; Paeratakul, O. Pharm. Res. 1989, 6, 413-417. (15) Illum, L.; Farraj, N. F.; Davis, S. S. Pharm. Res. 1994, 11, 11861189.
10.1021/la001754u CCC: $20.00 © 2001 American Chemical Society Published on Web 05/11/2001
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lenge.10 Contrary to the receptor-mediated internalization of most DNA-carrier complexes,16 uptake of these nanoparticles appears to occur even in the absence of any ligand-receptor interaction. Thus the major barrier in chitosan-DNA nanoparticle delivery is likely to be the effective passage across the cell membrane bilayer.10,17-20 In our recent biological study, it was determined that the incubation of pure chitosan with cells and the subsequent administration of chitosan-DNA nanoparticles altered the overall transfection efficiency (unpublished result). Moreover, the enhancement in transcellular transport of drugs across an intestinal epithelial monolayer21 induced by oral coadministrations of chitosan and drug led to our hypothesis that chitosan destabilized the cell membrane. Hence it is critical to understand the interaction between chitosan and cell membrane in order to optimize this novel strategy of gene and drug delivery. In this study, the fundamental interaction between chitosan and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane bilayer was probed by cross-polarization microscopy, differential scanning calorimetry (DSC), and Fourier transform (FT) Raman spectroscopy. Large multilamellar vesicles (MLV) of DPPC were chosen as our model system because DPPC is a major component of cell membranes and DPPC MLV has been widely used as a standard for characterizing cationic peptide,22 drug, and polymer induced membrane perturbations.23 In this study, DSC was used to elucidate the thermotropic behavior of DPPC-chitosan mixtures. Cross-polarization microscopy was applied to determine the structural features of MLV. The effect of hydrophobic driving force on the chitosanDPPC interaction was also addressed with a chloroform premixing step. FT-Raman spectroscopy holds the unparalleled capability of measuring molecular vibration spectrums of nucleic acids, lipids, proteins, and certain polymers.24-32 In detail, FT-Raman spectroscopy was applied to monitor the molecular orders of the hydrocarbon chains in the DPPC bilayer in the presence of chitosan. Our main goal in this investigation is to determine the role of pure chitosan in destabilizing the membrane for subsequent gene delivery of DNA-chitosan nanoparticles. (16) Varga, C. M.; Wickham, T. J.; Lauffenburger, D. A. Biotechnol. Bioeng. 2000, 70 (6), 593-605. (17) Chevallay, B.; Herbage, D. Med. Biol. Eng. Comput. 2000, 38 (2), 211-8. (18) Richardson, S. C.; Kolbe, H. V.; Duncan, R. Int. J. Pharm. 1999, 178 (2), 231-43. (19) MacLaughlin, F. C.; Mumper, R. J.; Wang, J.; Tagliaferri, J. M.; Gill, I.; Hinchcliffe, M.; Rolland, A. P. J. Controled Release 1998, 56 (1-3), 259-72. (20) Erbacher, P.; Zou, S.; Bettinger, T.; Steffan, A. M.; Remy, J. S. Pharm. Res. 1998, 15 (9), 1332-9. (21) Artursson, P.; Lindmark, T.; Davis S. S.; Illum, L. Pharm. Res. 1994, 11, 1358-1361. (22) Fresta, M.; Ricci, M.; Rossi, C.; Furneri, P. M.; Puglisi, G. J. Colloid Interface Sci. 2000, 226 (2), 222-230. (23) Osanai, S.; Nakamura, K. Biomaterials 2000, 21 (9), 867-876. (24) Sprunt, J. C.; Jayasooriya, U. A.; Wilson, R. H. Phys. Chem. Chem. Phys. 2000, 2 (19), 4299-4305. (25) Yuan, C. B.; Zhao, D. Q.; Zhao, B.; Wu, Y. J.; Liu, J. H.; Ni, J. Z. Langmuir 1996, 12 (22), 5375-5378. (26) Bouwstra, J. A.; Dubbelaar, F. E. R.; Gooris, G. S.; Weerheim, A. M.; Ponec, M. Biochim. Biophys. ActasBiomembrane 1999, 1419 (2), 127-136. (27) He, J. P.; You, N. T.; Chen, Q.; Hu, X. N.; Zheng, Q. X.; Li, S. P.; Squire, J. Acta Biochim. Biophys. Sin. 1999, 31 (2), 138-144. (28) Sailer, K.; Viaggi, S.; Nusse, M. Biochim. Biophys. Actas Biomembrane 1997, 1329 (2), 259-268. (29) Li, X. M.; Zhao, B.; Zhao, D. Q.; Ni, J. Z.; Wu, Y.; Xu, W. Q. Thin Solid Films 1996, 285, 762-764. (30) Pemberton, J. E.; Chamberlain, J. R. Biopolymers 2000, 57 (2), 103-116. (31) McCarthy, P. K.; Huang, C. H.; Levin, I. W. Biopolymers 2000, 57 (1), 2-10. (32) Batenjany, M. M.; OLeary, T. J.; Levin, I. W.; Mason, J. T. Biophys. J. 1997, 72 (4), 1695-1700.
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Experimental Section Materials. The dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in anhydrous powder form was obtained from Matryea Inc. (USA) and was used without further purification. Sodium chloride, sodium phosphate (monobasic), sodium phosphate (dibasic), potassium phosphate (monobasic), potassium phosphate (dibasic), potassium chloride, 1 N hydrochloric acid, and chloroform were obtained from Sigma Inc. (USA) and used as received. DNAease-free and 18 MΩ ultrapure water was obtained from Life Technologies Inc. (USA) and was used in the preparation of all solutions. Chitosan with a molecular weight of 113 kDa and degree of acetylation of 13% was obtained from Pronova Biomedical (Olso, Norway).10 Phosphate buffer saline (PBS) was prepared with 0.15 M sodium chloride, 0.01 M sodium phosphate, 0.05 M potassium chloride, and 0.08 M potassium phosphate and was adjusted to pH 7.4 with 1 N hydrochloric acid. Differential Scanning Calorimetry (DSC). DSC measurement was performed on a TA 2920 moderated DSC calorimeter (TA Instrument Inc., DE). Around 2 mg of DPPC anhydrous powder and 0-1.5 mg of chitosan were put in a hermetic DSC pan and mixed with 20 µL of PBS buffer. In another set of experiments, the mixture of DPPC and chitosan was first dissolved in chloroform, dried under nitrogen gas, and put in a vacuum desiccator overnight before rehydration in PBS buffer. For each DSC experiment, another sample containing 20 µL of plain PBS buffer in the DSC pan was used as a blank reference. Before the scan, the DSC pan was equilibrated at 60 °C with constant vortexing for at least 2 h in order to produce multilamellar vesicles (MLV)33,34 which was then checked by a crosspolarization microscope with detail procedures described elsewhere.35 The samples were scanned from 28 to 60 °C at a heating/ cooling rate of 0.5 °C/min for at least three times. The calorimetric enthalpy in the main transition and the corresponding phase transition temperature of the samples were determined with Universal Analysis Software (TA Instrument). The calculations of the Van’t Hoff enthalpy and cooperative unit (the ratio of Van’t Hoff and calorimetric enthalpy) were based on procedures described elsewhere.36,37 Fourier Transform (FT) Raman Spectroscopy. FT-Raman spectra were collected with a Nicole Nexus FT-IR spectrometer equipped with a FT-Raman module (Nicole Instruments, WI). The Raman samples of the DPPC (5 mg) mixed with various amounts of chitosan (0 or 4 mg) were first dispersed in 50 µL of PBS buffer and were equilibrated at 60 °C for at least 2 h under constant vortexing. The samples were then transferred to glass capillaries, sealed, and stored in a 4 °C refrigerator for at least 24 h before the experiment. The samples were excited with a continuous-wave diode pumped Nd:YAG of around 600 mW at the near-infrared spectrum of 1064 nm, and the backscattered emission from the excited sample was collected at a resolution of 4 cm-1 in the range of 1000-1200, 1200-1500, and 28003000 cm-1.
Results Figure 1a and Figure 1b show the cross-polarization images of pure DPPC MLV and DPPC/chitosan mixture (chitosan mole fraction, 0.29%) after hydration at 60 °C. The average size of DPPC MLV was increased from 20 to 50 µm when chitosan was added. Figure 2a shows the DSC thermograms of DPPC/chitosan/water system with the mole fraction of chitosan to DPPC ranging from 0 to 0.49% during DSC heating scan. Pure DPPC bilayer underwent gel to liquid crystalline transition at around (33) Savva, M.; Torchilin, V. P.; Huang, L. J. Colloid Interface Sci. 1999, 217, 160-165. (34) Kasbauer, M.; Junglas, M.; Bayerl, T. M. Biophys. J. 1999, 76, 2600-2605. (35) Kiyoshi, M.; Koichi, S.; Kiyomitsu, S. Colloids Surf., B 1997, 9, 9-15. (36) Johann, C; Garidel, P; Mennicke, L; Blume, A. Biophys. J. 1996, 71 (6), 3215-3228. (37) Koberl, M.; Schoppe, A.; Hinz, H. J.; Rapp, G. Chem. Phys. Lipids 1998, 95 (1), 59-82.
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Figure 1. (a, top) Cross-polarization images of the large multilamellar vesicles of pure DPPC. (b, bottom) Cross-polarization images of a DPPC/chitosan mixture (chitosan mole fraction, 0.29%).
39.5 °C. As the mole fraction of chitosan increased from 0 to 0.49%, the peak height of the main phase transition of hydrated DPPC bilayer successively decreased. In contrast, the temperature at the gel to liquid crystalline transition of DPPC (Tm) was independent of chitosan mole fraction and stayed at around 39.5 °C. Moreover, a pretransition peak was detected on each endotherm at around 33 °C. Figure 2b shows the total calorimetric
enthalpy of the main phase transition and pretransition against chitosan mole fraction in chitosan/DPPC/water mixtures during the DSC heating scans. The calorimetric enthalpy of pure DPPC in the phase transition was 9.1 kcal/mol. At low chitosan mole fraction of 0.08%, the calorimetric enthalpy of the phase transition became 8.4 kcal/mol. At the highest chitosan concentration of 0.49%, the calorimetric enthalpy was reduced to 4.4 kcal/mol.
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Figure 2. (a) DSC thermograms of a DPPC/chitosan/water system with chitosan mole fraction ranging from 0 to 0.49% during sample heating from 25 to 60 °C. (b) Endothermic enthalpy of the observed phase changes (the total enthalpy of both main phase transition and pretransition) against the mole fraction of chitosan in the chitosan/DPPC/water mixture during the DSC heating scans.
Figure 3a shows the DSC cooling thermograms of the hydrated DPPC bilayer at different chitosan mole fractions. Each cooling thermogram had a similar trend and showed a single exothermic transition. As chitosan mole fraction increased from 0 to 0.49%, the peak of the exothermic transition from liquid crystalline to gel phase was successively depressed. Figure 3b showed the calorimetric enthalpy in the phase transition of DPPC during the DSC cooling scans. In pure DPPC bilayer, the calorimetric enthalpy was 9.1 kcal/mol. At chitosan mole fraction of 0.08%, the calorimetric enthalpy was reduced to 6.5 kcal/mol. Overall, the slope of the calorimetric enthalpy vs chitosan concentration curve during cooling was larger than that of the heating scans. At chitosan mole fraction of 0.49%, the calorimetric enthalpy was reduced by more than two-thirds to 2.9 kcal/mol. Figure 4a shows a series of DSC endotherms of DPPC/ chitosan/water mixtures that were predissolved in chloroform and dried before hydration. An increase of chitosan mole fraction resulted in significant reduction in the peak height of the endothermic transition. Furthermore, the width of the endothermic peak increased when chitosan mole fraction increased. Figure 4b showed the change of endothermic enthalpy in DPPC phase transition for the chloroform premixed samples against the chitosan mole fraction during sample heating. In pure DPPC predissolved in chloroform, the endothermic enthalpy was 8.5 kcal/mol. The calorimetric enthalpy of the DPPC phase transition decreased as the amount of chitosan increased. At chitosan mole fraction of 0.08%, the endothermic enthalpy was dramatically reduced to 4.7 kcal/mol. The
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Figure 3. (a) DSC thermograms of the hydrated DPPC bilayer during sample cooling in the presence of different chitosan mole fractions. (b) Exothermic enthalpy in the phase transition of DPPC against chitosan mole fraction during the DSC cooling scans.
endothermic enthalpy reached a plateau of about 4 kcal/ mol in the higher concentration regime of chitosan. Figure 5a shows the DSC thermograms of DPPC that was premixed with various mole fractions of chitosan in chloroform before hydration during sample cooling. Pure DPPC had the highest peak among the four exotherms and contained one exothermic transition. The amplitude of the exothermic peak generally decreased with the chitosan mole fraction. At a chitosan mole fraction of 0.29%, a new exothermic peak was detected at 33 °C. Most interestingly, a new endothermic peak at 35 °C was detected at the highest chitosan mole fraction of 0.49%. Figure 5b showed the calorimetric enthalpy of DPPC/ chitosan/water mixture (chloroform premixed) against chitosan mole fraction during sample cooling. An increase of chitosan mole fraction suppressed the phase transition enthalpy of the DPPC bilayer. The slope of the curve was highest at the lower concentration regime of chitosan and then remained constant from 1 to 6.4 wt %. At the highest chitosan mole fraction of 0.49%, the exothermic enthalpy dropped by 30% to 2.1 kcal/mol from that of pure DPPC previously dissolved in chloroform. Figure 6 showed the plot of cooperative unit against chitosan mole fraction for both sets of samples that were either mixed in dry powder form or premixed in chloroform. Overall, an increase of chitosan content led to a significant decrease of the cooperative unit of both types of samples. The cooperative unit of dry and chloroform predissolved pure DPPC were 171 and 167, respectively. However, the reduction rate of the cooperative unit against the chitosan mole fraction was an order of magnitude higher in the chloroform predissolved samples. At the lowest chitosan mole fraction of 0.08%, the cooperative unit of dry mixed
Membrane Perturbation with Chitosan
Figure 4. (a) A series of endotherms of a DPPC/chitosan/water mixture (with different chitosan mole fractions) that was predissolved in chloroform and dried before taking the DSC heating scans. (b) Endothermic enthalpy in DPPC phase transition for the chloroform premixed samples against the chitosan mole fraction during the sample heating.
sample and chloroform premixed sample was 167 and 46, respectively. At the highest chitosan mole fraction of 0.49%, the cooperative unit of dry and chloroform premixed DPPC-chitosan reached levels of 54 and 23, respectively. Figure 7a shows the FT-Raman spectra of DPPC powder, a hydrated DPPC bilayer, and a hydrated DPPCchitosan mixture in the lower vibrational energy regime of 1000-1200 cm-1. Three clear peaks were detected at 1062, 1099, and 1128 cm-1 on all the Raman spectra of all three samples. The ratios of peak height intensity are shown in Table 1. The ratio of peak height intensity at 1099 and 1062 cm-1 was highest with a value of 0.83 in hydrated DPPC/chitosan/water mixture and was lowest with a value of 0.65 in pure DPPC powder (sodium salt). The I1099/I1128 ratios of chitosan/DPPC/water, DPPC/water, and DPPC anhydrous powder were 0.98, 0.97, and 0.91, respectively. Figure 7b shows the Raman spectra of the three samples from 1200 to 1500 cm-1. The ratios of the peak intensity at 1460 and that in 1436 cm-1 in all three samples were all around 0.85. Figure 7c shows the Raman spectra of the three samples from 2800 to 3000 cm-1. Distinguishable peaks were detected on all three Raman spectra at the vibrational frequencies of 2848, 2880, and 2932 cm-1. Table 1 summarizes all the Raman intensity ratios of I2848/I2880 and I2932/I2880 for the three samples. The ratio of I2932/I2880 was highest with a value of 0.96 in chitosan/DPPC/water mixture and was lowest with a value of 0.43 in DPPC powder.
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Figure 5. (a) DSC thermograms of DPPC that was premixed with various mole fractions of chitosan in chloroform before hydration during sample cooling. (b) Exothermic enthalpy of DPPC/chitosan/water mixture (chloroform premixed) against the chitosan mole fraction during DSC cooling scans.
Figure 6. Cooperative unit against the mole fraction of chitosan for both sets of samples that were either mixed in aqueous solution or premixed in chloroform before hydration.
Discussions DSC has been recently applied to elucidate the interactions of poly(vinylpyrrolidone) (PVP),33 peptide,22 and sugar38 with large multilamellar vesicles (MLV) of DPPC. Recently, one group has provided experimental evidence on the complex formation between chitosan and DPPC (38) Nagase, H.; Ueda, H.; Nakagaki, M. Biochim. Biophys. Acta 1997, 1328, 197-206.
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Table 1. Effect of Chitosan (MW, 113 kDa) on the Ratio of Raman Peak Height Intensity of DPPC
anhydrous DPPC powder hydrated DPPC bilayer hydrated DPPC bilayer + chitosan
I(1099 cm-1)/ I(1062 cm-1)
I(1099 cm-1)/ I(1128 cm-1)
I(1460 cm-1)/ I(1436 cm-1)
I(2848 cm-1)/ I(2881 cm-1)
I(2932 cm-1)/ I(2880 cm-1)
0.65 0.76 0.83
0.91 0.97 0.98
0.87 0.83 0.81
0.72 0.53 0.55
0.43 0.89 0.96
Figure 7. The FT-Raman spectra of DPPC powder, hydrated DPPC bilayer, and hydrated DPPC/chitosan mixture.: (a) lower vibrational energy regime from 1000 to 1200 cm-1; (b) middle vibrational energy regime from 1200 to 1500 cm-1; (c) higher vibrational energy regime from 2800 to 3000 cm-1.
around oil droplets in an oil-in-water emulsion.39 However, the interaction between chitosan and the MLV of DPPC in aqueous solution has not been reported in the context of bilayer membrane perturbation. This type of study is crucial because our group has recently found that chitosan acts as a cell membrane perturbant for the following (39) Magdassi, S.; Bach, U.; Mumcuoglu, K. Y. J. Microencapsulation 1997, 14 (2), 189-95.
delivery of chitosan-DNA particles. Chitosan becomes a polycation when its primary amines (degree of acetylation was 13%) are protonated at a pKa of 6.2-7.40 At the same time, the presence of N-acetyl groups (-COCH3) on the chitosan backbone imparts hydrophobic properties in addition to the characteristics of a soluble polycation.40 More important, the high ionic strength and significant degree of acetylation of chitosan used in this study would result in a combined electrostatic-hydrophobic driving force for chitosan-induced membrane destabilization. In this study, the existence of DPPC MLV was proved by the cross-polarization image of the small DPPC vesicles. It appeared that chitosan solution surrounding the MLV of DPPC was sufficient to induce DPPC-chitosan interaction as shown by the fusion of MLV to form bigger vesicles. One group reported that DPPC vesicle fusion originated from the bilayer destabilization caused by membrane perturbant such as fusion peptide.41 The DPPC bilayer perturbation caused by chitosan also supported our recent observation that pure chitosan destabilized cell membranes for the subsequent delivery of chitosanDNA complex (unpublished result). In addition, chitosan fibers or sheets were not formed under the current experimental conditions. The prerequisite of premixing polymer and DPPC in chloroform reported in several studies was not required for DPPC-chitosan interaction.33,42 For example, poly(vinylpyrrolidone) (PVP) did not interact with the DPPC MLV in aqueous solution as shown by DSC measurement and photon correlation spectroscopy.33 In general, a shift of the phase transition temperature Tm and/or calorimetric enthalpy in hydrated DPPC bilayer signifies changes in the bilayer organization. Our measured Tm and total enthalpy of 39.5 °C and 9.05 kcal/mol of the pure DPPC transformation from Lβ′ to LR phase agreed well with the reported values of 41 °C and 9.1 kcal/mol, respectively, for DPPC MLV.33,34,36,38 The presence of one main endothermic peak in the gel-liquid crystalline transition proved that chitosan did not induce phase separation on DPPC bilayer. It is because phase separations of the DPPC bilayer induced by the polymer were characterized by the splitting of the main endothermic peak into two or more peaks.43 In general, a change in phase transition enthalpy without any change in Tm as seen in the DPPC/chitosan interaction was classified as an “A”-type change.22 This type of transformation indicates the localization of foreign molecules in the outer cooperative zone of the bilayer.22 Also, the phase transition enthalpy highly depends on the interactions between individual lipid molecules in the bilayer.38 Similar concentration dependency of phase transition enthalpy of DPPC bilayer was reported in the DPPC/lung surfactants interactions.44 In addition, a cooperative unit allows the estimation of the number of (40) Ratto, J.; Hatakeyama, T.; Blumstein, R. B. Polymer 1995, 36 (15), 2915-2919. (41) Longo, M. L.; Waring, A. J.; Hammer, D. A. Biophys. J. 1997, 73 (3), 1430-1439. (42) Johann, C.; Garidel, P.; Mennicke, L.; Blume, A. Biophys. J. 1996, 71, 3215-3228. (43) Nakano, M.; Inoue, R.; Koda, M.; Baba, T.; Matsunaga, H.; Natori, T.; Handa, T. Langmuir 2000, 16, 7156-7161. (44) Shiffer, K.; Hawgood, S.; Haagsman, H. P.; Benson, B.; Clements, J. A.; Goerke, J. Biochemistry 1993, 32 (2), 590-597.
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DPPC molecules undergoing the gel-liquid crystalline transition simultaneously.36,37 Pure DPPC MLV dispersions have a high cooperative unit during the main phase transition (0% mole fraction).37 The strong interactions between the DPPC bilayer and associative foreign molecules always reduce the number of molecules undertaking simultaneous phase transition and drive down the cooperative unit.33,36,37 At all chitosan mole fractions, chitosan association on the DPPC bilayer surface caused serious disorganization on the ordered bilayer structure and resulted in significant reduction of the cooperative unit. One group hypothesized that the reductions of cooperative unit and enthalpy in cationic peptide/DPPC MLV mixtures originated from the electrostatic repulsion between the protonated amine on the peptide and the positively charged choline headgroups of DPPC in a high ionic strength solution.22 This hypothesis was supported by the further reduction of enthalpy in acidic solution when most primary amines of chitosan were protonated (data not shown). The phase transition of a DPPC bilayer was generally regarded as a reversible process.43,44 The major difference between sample heating and cooling was the disappearance of the pretransition peak. The presence of a single peak in the main phase transition during sample cooling reassured that no new phase was formed on the DPPC bilayer and the main transition was generally reversible when chitosan and DPPC were mixed in dry powder form. The absence of phase transition from the rippled-like Pβ to the stabilized LR phase around 33 °C in DPPC during cooling was shown in numerous studies.33,36,44,45 In pure DPPC, the exothermic enthalpy of main transition was close to the endothermic enthalpy during sample heating.29 Chitosan had a more pronounced effect on the gel-liquid crystalline transition of DPPC during sample cooling as shown by the larger slope on the enthalpy vs chitosan mole fraction plot. Furthermore, the exothermic enthalpy was 30% smaller than the endothermic enthalpy at the highest chitosan mole fraction of 0.49%. The same trend was reproducible during successive DSC heating/cooling scans of the DPPC/chitosan/water systems (data not shown). The result implied that chitosan associated more effectively with DPPC bilayer in the liquid crystalline phase. The formation of larger MLV in the presence of chitosan (as shown in cross-polarized images) indicated that the bilayer stability of pure DPPC MLV was further reduced by chitosan after sample heating at 60 °C. Chloroform has been used as a standard solvent for enhancing the hydrophobic interaction of DPPC bilayer with other lipids,43 polymers,33,44 peptide,22 and drugs.42,46 The extent of DSC peak broadening was more obvious in chloroform mixed samples than the direct hydrated samples and indicated the increased miscibility between chitosan and DPPC. In principle, the peak broadening in the DSC thermograms was caused by the variation among different populations of lipid molecules across the bilayer structure and was often found in well-mixed foreign/DPPC mixtures.33,36,37 The enhancement in DPPC/chitosan miscibility and lipid-polymer interactions was further supported by the amplified reductions of endothermic enthalpy and cooperative unit against increasing chitosan mole fraction during sample heating. This result was likely caused by the increased hydrophobic interaction between the acetyl groups of chitosan and the acyl chain of DPPC (45) Rappolt, M.; Pabst, G.; Rapp, G.; Kriechbaum, M.; Amenitsch, H.; Krenn, C.; Bernstroff, S.; Laggner, P. Eur. Biophys. J. Biophys. Lett. 2000, 29 (2), 125-133. (46) Wojtowicz, K.; Gruszecki, W. I.; Walicka, M.; Barwicz, J. Biochim. Biophys. ActasBiomembrane 1998, 1373 (1), 220-226.
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when they are mixed in a nonpolar solvent as shown in cationic peptide/DPPC22 and PVP/DPPC interactions.33 During cooling of chloroform premixed DPPC/chitosan mixtures, obscure features in the cooling thermograms were apparent at higher chitosan mole fractions of 0.29 and 0.49%. The splitting of one single exothermic peak into two peaks at 0.29% indicated that two distinct phases were formed on the bilayer during sample cooling and was commonly found in the interactions of polymer, drugs, and peptides with DPPC MLV.36,37,43-46 Obviously, the main transition peak that remained at 39.5 °C corresponded to pure DPPC bilayer. The smaller peak at around 33 °C was rarely detected during DPPC MLV cooling36,37 and was likely caused by the chitosan-induced destabilization of the DPPC bilayer. The membrane disruption was supported by the MLV fusions observed under cross-polarization microscopy. At a chitosan mole fraction of 0.49%, the single exothermic transition was split into one major exothermic transition peak at 39.5 °C and a minor endothermic transition peak at 35 °C. The detection of this new endothermic peak provided further evidence of the effect of chitosan in destabilizing the membrane bilayer. In pure chitosan solution, the only detectable phase was an extremely weak glass transition at 27 °C and the transition enthalpy was around 2 J/g.40 Thus these new phases were not caused by chitosan alone and it must have involved the interactions between chitosan and DPPC bilayer. Several studies have shown that Raman spectroscopy elucidated the structural organization of DPPC MLV under the influences of foreign molecules.24-32,47 In this study, only three distinct samples of pure DPPC, DPPC/ water mixture, and DPPC/chitosan/water mixture (chitosan mole fraction, 0.49%) were focused in order to demonstrate the overall effect of chitosan on the molecular properties of DPPC bilayer. The high concentration of chitosan used in our FT-Raman measurement should give solid fingerprints on the nature of chitosan-DPPC interaction. The completed Raman spectra of DPPC in the three vibrational regimes of 1000-1200, 1200-1500, and 2800-3000 cm-1 were simultaneously reported. In the lowest vibrational range, the peaks at 1062, 1099, and 1128 cm-1 represented the C-C stretching vibrations of all-trans segments, C-C stretching vibrations of the gauche conformer, and C-C stretching vibrations of trans conformer, respectively, in the acyl chains inside the bilayer.26,29,31 The peak intensity ratios of I1099/I1062 and I1099/I1128 have been used to quantify the amount of gauche bonds relative to the amount of trans bonds and to reveal the bilayer organizations in several studies of membraneforeign molecules interactions.26-32 I1099/I1062 and I1099/I1128 of DPPC bilayer were increased from 0.76 to 0.83 and from 0.97 to 0.98, respectively, with the addition of chitosan. The increase of two ratios indicated that there was a rise of gauche/trans population and proved that the interchain order of the acyl groups decreased. More important, this result suggested that chitosan increased the membrane fluidity and the DPPC-chitosan complex was more disordered than pure DPPC bilayer. In addition, the two ratios of anhydrous DPPC powder are also shown in Table 1 as a reference because it was hard to make a direct comparison between an anhydrous DPPC solid and hydrated DPPC bilayer. DPPC powder has the lowest values in I1099/I1062 and I1099/I1128 of 0.65 and 0.91, respectively, among the three samples as the interchain order of DPPC molecules in solid state was higher than that in (47) Huang, H.; Lapides, J. R.; Levin, I. W. J. Am. Chem. Soc. 1982, 104, 5926.
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the hydrated bilayer as shown by the studies of other lipid powders.27,29,31 At this lower vibrational range, the results showed that chitosan-induced destabilization of DPPC MLV originated from strong intermolecular interactions. The deformation of the methylene group (CH2) was found in the spectral regime of 1200-1400 cm-1 and has been used to characterize lipid lateral packing.29,30,41,42,46 The peaks at 1460 and 1436 cm-1 represent the highfrequency component of CH2 deformation and lowfrequency component of CH2 deformation, respectively. A change of I(1460 cm-1)/I(1436 cm-1) ratio can be used for elucidating lipid interchain interactions. Our results showed that chitosan-DPPC interaction did not cause any detectable change in the CH2 deformation. In the highest vibrational regime, the peaks at 2848, 2880, and 2932 cm-1 represent the methylene C-H symmetric stretching, the methylene C-H antisymmetric stretching, and Fermi interaction of the methylene groups with the terminal methyl group, respectively, in the acyl chains of the DPPC bilayer.29,30,39-43,45,46 The I2848/I2880 intensity ratio has been widely used for characterizing the lateral chainchain interactions that dictate the organization of the bilayer and an increase of the ratio indicated a rise of intermolecular disorder of the acyl chain.27,29,30,41-43,46 Simultaneously, the I2932/I2880 intensity ratio was widely used for monitoring the stability of the intramolecular conformation of the acyl chains in the bilayer, and an increase of the ratio was regarded as an increase of intrachain disorder in DPPC molecules. I2848/I2880 and I2932/I2880 of DPPC bilayer and DPPC/ chitosan mixture were increased from 0.53 to 0.55 and from 0.89 to 0.96, respectively, when chitosan interacted with DPPC MLV. The increase of the two ratios indicated that chitosan-DPPC interactions directly influenced the lateral chain interaction and induced disorder in the packing of the acyl chain. This result implied that chitosan could penetrate into the acyl chains of the cell membrane
Chan et al.
bilayer and proved that chitosan significantly disrupted the membrane organization as shown by our other results. The chitosan-induced destabilization was confirmed by the higher initial release rate of quinine release from liposome/chitosan complex in comparison with pure liposomes.48 DPPC powder has the lowest I2932/I2880 among the three samples as the intramolecular order of DPPC in solid state was higher than that in hydrated bilayer and agreed with other reported values.25,47 Conclusion In summary, chitosan, which was proven as a potential membrane perturbant for subsequent gene delivery of chitosan-DNA nanoparticles in cellular studies, interacted strongly with a standard membrane bilayer. The strong interactions led to a decrease of phase transition enthalpy and a reduction of cooperative unit during the main transition of the DPPC bilayer. The membrane perturbation caused by chitosan was confirmed by crosspolarization microscopy. The enhancement of hydrophobic interaction between chitosan and DPPC in chloroform resulted in further destabilization of the DPPC MLV. The FT-Raman spectroscopy study revealed that chitosan tailored the intermolecular and intramolecular interactions of the lipid molecules in the bilayer. This study provided valuable physical insights into the mechanism of chitosan-induced perturbation of a model membrane. Acknowledgment. Two authors (M.H. and K.W.L.) were supported by a NIH Grant (5P01CA79862-020003) and by Johns Hopkins Singapore, Inc. One author (V.C.) thanks Dr. Sandy Chian (NTU) for allowing him to use the facilities of his laboratory and was supported by NTU AcRF fund RG 15/00. LA001754U (48) Henriksen, I.; Vagen, S. R.; Sande, S. A.; Smistad, G.; Karlsen, J. Int. J. Pharm. 1997, 146 (2), 193-203.
